Improving crop tolerance to heavy metal stress by polyamine application.

Many areas have been heavily contaminated by heavy metals from industry and are not suitable for food production. The consumption of contaminated foods represents a health risk in humans, although some heavy metals are essential at low concentrations. Increasing the concentrations of essential elements in foods is one goal to improve nutrition. The aim of this study was to increase the accumulation of heavy metals in plant foods by the external application of putrescine. The levels of cadmium, zinc and iron were measured in different vegetables grown in hydroponic medium supplemented with heavy metals and compared with those grown in a reference medium. The estimated daily intake, based on the average daily consumption for various vegetable types, and the influence of polyamines on metal uptake were calculated.

Polyamine transporters and polyamines increase furfural tolerance during xylose fermentation with ethanologenic Escherichia coli strain LY180.

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.

Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells.

The redox environment of the cell is currently thought to be extremely important to control either apoptosis or autophagy. This study reported that reactive oxygen species (ROS) and nitric oxide (NO) generations were induced by evodiamine time-dependently; while they acted in synergy to trigger mitochondria-dependent apoptosis by induction of mitochondrial membrane permeabilization (MMP) through increasing the Bax/Bcl-2 or Bcl-x(L) ratio. Autophagy was also stimulated by evodiamine, as demonstrated by the positive autophagosome-specific dye monodansylcadaverine (MDC) staining as well as the expressions of autophagy-related proteins, Beclin 1 and LC3. Pre-treatment with 3-MA, the specific inhibitor for autophagy, dose-dependently decreased cell viability, indicating a survival function of autophagy. Importantly, autophagy was found to be promoted or inhibited by ROS/NO in response to the severity of oxidative stress. These findings could help shed light on the complex regulation of intracellular redox status on the balance of autophagy and apoptosis in anti-cancer therapies.

Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements.

A number of studies show that environmental stress conditions such as drought, high salt, and air pollutants increase polyamine levels in plant cells. However, little is understood about the physiological function of elevated polyamine levels. We report here that polyamines regulate the voltage-dependent inward K(+) channel in the plasma membrane of guard cells and modulate stomatal aperture, a plant "sensor" to environmental changes. All natural polyamines, including spermidine, spermine, cadaverine, and putrescine, strongly inhibited opening and induced closure of stomata. Whole-cell patch-clamp analysis showed that intracellular application of polyamines inhibited the inward K(+) current across the plasma membrane of guard cells. Single-channel recording analysis indicated that polyamine regulation of the K(+) channel requires unknown cytoplasmic factors. In an effort to identify the target channel at the molecular level, we found that spermidine inhibited the inward K(+) current carried by KAT1 channel that was functionally expressed in a plant cell model. These findings suggest that polyamines target KAT1-like inward K(+) channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine levels, and stomatal regulation.

Characterization of a macrophage-based system for studying the activation of latent TGF-beta.

TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF-beta activation, thus providing a screening method for potential anti-scarring molecules.

Regulation of plant cell-wall pectin methyl esterase by polyamines--interactions with the effects of metal ions.

The kinetic study of the de-esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell-shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell-wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell-wall enzymes are involved.

Newly synthesized analogues of the spider toxin block the crustacean glutamate receptor.

Effects of synthetic compounds similar to the structure of a spider toxin were studied on the glutamate receptors in crustacean neuromuscular synapses. Two kinds of analogues, 2,4-dihydroxyphenylacetyl-asparaginyl cadaverine (C-1) and 2,4-dihydroxyphenylacetyl-asparaginyl spermine (C-2), suppressed the excitatory postsynaptic potentials in a manner similar to natural spider toxin (JSTX). The dose-response relationship showed that the relative potency of the compounds is C-1 less than C-2 less than JSTX. While the effect of JSTX was irreversible, those of C-1 and C-2 were reversible. These synthetic compounds may serve as important tools in studying the structure and function of glutamate receptors.

Effects of intracerebroventricular administration of aliphatic diamines on ingestive behavior in the rat.

We examined the pharmacological effects of intracerebroventricularly administered aliphatic diamines on ingestive behavior in male rats adapted to a 4 hr per day feeding and drinking schedule. 1,2-Ethanediamine (ETD), 1,3-propanediamine (PRD), 1,4-butanediamine (putrescine, PUT), 1,5-pentanediamine (cadaverine, CAD) and 1,6-hexanediamine (HED) suppressed feeding and drinking behavior in a dose-dependent manner, but not unless a relatively high dose (over 80 micrograms) was given. The approximate anorectic potency was HED greater than CAD divided by PUT greater than ETD greater than PRD. A sedation was also produced in fairly good parallel to these alterations in feeding and drinking behavior. Thus, there appears to be a relationship between the length of the carbon chain and the potency of the pharmacological action, and these inhibitory effects on feeding and drinking behavior are probably not due to a specific action on the regulatory system for ingestive behavior, but rather to a nonspecific action.

Enzymatic methylations: III. Cadaverine-induced conformational changes of E. coli tRNA fMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation.

A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.